1. Field Of the Invention
This invention relates to amino acid transporters from mammalian species and the genes corresponding to such transporters. Specifically, the invention relates to the isolation, cloning and sequencing of complementary DNA (cDNA) copies of messenger RNA (mRNA) encoding each of four novel human amino acid transporter genes. The invention also relates to the construction of recombinant expression constructs comprising such cDNAs from each of the four novel human amino acid transporter genes of the invention, said recombinant expression constructs being capable of expressing amino acid transporter proteins in cultures of transformed prokaryotic and eukaryotic cells. Production of the transporter proteins in such cultures is also provided. The invention relates to the use of such cultures of such transformed cells to produce homogeneous compositions of each transporter protein. The invention also provides cultures of such cells producing transporter proteins for the characterization of novel and useful drugs. Antibodies against and epitopes of these transporter proteins are also provided by the invention.
2. Background of the Invention
The approximately 20 naturally-occurring amino acids are the basic building blocks for protein biosynthesis. Certain amino acids, such as glutamate and glycine, as well as amino acid derivatives such as .gamma.-aminobutyric acid (GABA), epinephrine and norepinephrine, and histamine, are also used as signaling molecules in higher organisms such as man. For these reasons, specialized trans-membrane transporter proteins have evolved in all organisms to recover or scavenge extracellular amino acids (see Christensen, 1990, Physiol. Rev. 70: 43-77 for review).
These transporter proteins play a particularly important role in uptake of extracellular amino acids in the vertebrate brain (see Nicholls & Attwell, 1990, TiPS 11: 462-468). Amino acids that function as neurotransmitters must be scavenged from the synaptic cleft between neurons to enable continuous repetitive synaptic transmission. More importantly, it has been found that high extracellular concentrations of certain amino acids (including glutamate and cysteine) can cause neuronal cell death. High extracellular amino acid concentrations are associated with a number of pathological conditions, including ischemia, anoxia and hypoglycemia, as well as chronic illnesses such as Huntington's disease, Parkinson's disease, Alzheimer's disease, epilepsy and amyotrophic lateral sclerosis (ALS; see Pines et al., 1992, Nature 360: 464-467).
Glutamate is one example of such an amino acid. Glummate is an excitatory neurotransmitter (i.e., excitatory neurons use glutamate as a neurotransmitter). When present in excess (&gt;about 300 .mu.M; Bouvier et al., 1992, Nature 360: 471-474; Nicholls & Attwell, ibid.; &gt;5 .mu.M for 5 min.; Choi et al., 1987, J. Neurosci. 7: 357-358), extracellular glutamate causes neuronal cell death. Glummate transporters play a pivotal role in maintaining non-toxic extracellular concentrations of glutamate in the brain. During anoxic conditions (such as occur during ischemia), the amount of extracellular glutamate in the brain rises dramatically. This is in part due to the fact that, under anoxic conditions, glutamate transporters work in reverse, thereby increasing rather than decreasing the amount of extracellular glutamate found in the brain. The resultingly high extracellular concentration of glutamate causes neuron death, with extremely deleterious consequences for motor and other brain functions, resulting in stroke, anoxia and other instances of organic brain dysfunction.
This important role for amino acid transporters in maintaining brain homeostasis of extracellular amino acid concentrations has provided the impetus for the search for and development of compounds to modulate and control transporter function. However, conventional screening methods require the use of animal brain slices in binding assays as a first step. This is suboptima/for a number of reasons, including interference in the binding assay by non-specific binding of heterologous (i.e., non-transporter) cell surface proteins expressed by brain cells in such slices; differential binding by cells other than neuronal cells present in the brain slice, such as glial cells or blood cells; and the possibility that putative drug binding behavior in animal brain cells will differ from the binding behavior in human brain cells in subtle but critical ways. The ability to synthesize human transporter molecules in vitro would provide an efficient and economical means for rational drug design and rapid screening of potentially useful compounds.
Amino acid transporters are known in the art, and some of these proteins have been isolated biochemically and their corresponding genes have been recently cloned using genetic engineering means.
Christensen et al., 1967, J. Biol. Chem. 242:5237-5246 report the discovery of a neutral amino acid transporter (termed the ACS transporter) in Erlich ascites tumor cells.
Makowske & Christensen, 1982, I. Biol. Chem. 257:14635-14638 provide a biochemical characterization of hepatic amino acid transport.
Kanner & Schuldiner, 1987, CRC Crit. Rev. Biochem. 22:1-38 provide a review of the biochemistry of neurotransmitters.
Olney et al., 1990, Science 248:596-599 disclose that the amino acid cysteine is a neurotoxin when present in excess extracellularly.
Wallace et al., 1990, J. Bacteriol. 172:3214-3220 report the cloning and sequencing of a glutamate/aspartate transporter gene termed gltP from Escherichia coli strain K12.
Kim et al., 1991, Nature 352:725-728 report the discovery that a cationic amino acid transporter is the cell surface target for infection by ecotropic retroviruses in mice.
Wang et al., 1991, Nature 352:729-731 report the discovery that a cationic amino acid transporter is the cell surface target for infection by ecotropic retroviruses in mice.
Maenz et al., 1992, J. Biol. Chem. 267:1510-1516 provide a biochemical characterization of amino acid transport in rabbit jejunal brush border membranes.
Bussolati et al., 1992, J. Biol. Chem. 267:8330-8335 report that the ASC transporter acts in an electrochemically neutral manner so that sodium ion co-transport occurs without disrupting the normal membrane potential of the cells expressing the transporter.
Engelke et al., 1992, J. Bacteriol. 171:5551-5560 report the cloning of a dicarboxylate carrier from Rhizobium meliloti.
Guastella et al., 1992, Proc. Natl. Acad. Sci. USA 89:7189-7193 disclose the cloning of a sodium ion and chloride ion-dependent glycine transporter from a glioma cell line that is expressed in the rat forebrain and cerebellum.
Kavanaugh et al., 1992, J. Biol. Chem. 267:22007-22009 report that biochemical characterization of a rat brain GABA transporter expressed in vitro in Xenopus laevis oocytes.
Storck et al., 1992, Proc. Natl. Acad. Sci. USA 89:10955-10959 disclose the cloning and sequencing of a sodium ion-dependent glutamate/aspartate transporter from rat brain termed GLAST1.
Bouvier et al., ibid., disclose the biochemical characterization of a glial cell-derived glutamate transporter.
Pines et al., ibid., report the cloning and sequencing of a glial cell glutamate transporter from rat brain termed GLT-1.
Kanal & Hediger, 1992, Nature 360:467-471 disclose the cloning and sequencing of a sodium ion-dependent, high affinity glutamate transporter from rabbit small intestine termed EAAC1.
Kong et at., 1993, J. Biol. Chem. 268:1509-1512 report the cloning and sequencing of a sodium-ion dependent neutral amino acid transporter of the A type that is homologous to a sodium-ion dependent glucose transporter.
Nicholls & Attwell, ibid., review the role of amino acids and amino acid transporters in normal and pathological brain functions.